The recommended dietary allowance for vitamin C (ascorbate) of 60mg daily is based on preventing the deficiency disease scurvy. We proposed that a new recommended dietary allowance for vitamin C can be determined using the principles of in situ kinetics. In situ kinetics has biochemical and clinical components. The goals of the biochemical components are to determine vitamin C molecular functions in relation to vitamin concentrations. For these studies vitamin C is investigated in human tissues such as fibroblasts and neutrophils. To determine how intracellular concentration is regulated, two mechanisms of vitamin transport were characterized. Ascorbate is transported by carriers which are sodium-dependent, saturable, energy dependent, and inhibited by newly synthesized ascorbate analogs. In contrast, oxidized vitamin C is transported by glucose transporters GLUT I, III, and IV, and immediately reduced intracellularly to ascorbate. The protein responsible for reduction in neutrophils was isolated, sequenced, identified as thioltransferase (glutaredoxin), and cloned. The full length human glutaredoxin gene was cloned and its promoter characterized. These studies indicate that vitamin C function in neutrophils may be to protect neutrophils from their own oxidants. Overall findings suggest that vitamin C function can be determined in relation to its concentration in living tissues. The clinical components of in situ kinetics are to determine how vitamin concentrations are achieved in normal humans as a function of dose and whether concentrations humans achieve are those which regulate molecular functions of the vitamin. A clinical trial was completed in healthy male inpatients hospitalized at the Clinical Center. For the first time, the following were described: the relationship between vitamin C doses over a wide range and its concentration in plasma and tissues; true bioavailability of vitamin C; vitamin urinary excretion in relation to dose; and potential adverse effects in relation to dose. A clinical trial in healthy women is underway. Based on biochemical and clinical evidence, we propose that a new recommended dietary allowance for vitamin C in healthy men is at least 100mg, and preferably 200 mg, daily. These findings may influence dietary guidelines for vitamin C now being prepared by the National Academy of Sciences. - Vitamin C, Nutrition, Ingestion Recommendations - Human Subjects